Antimicrobial Resistance and Mechanisms of Azithromycin Resistance in Nontyphoidal Salmonella Isolates in Taiwan, 2017 to 2018

ABSTRACT Antimicrobial resistance was investigated in 2,341 nontyphoidal Salmonella (NTS) isolates recovered from humans in Taiwan from 2017 to 2018 using antimicrobial susceptibility testing. Azithromycin resistance determinants were detected in 175 selected isolates using PCR and confirmed in 81 selected isolates using whole-genome sequencing. Multidrug resistance was found in 47.3% of total isolates and 96.2% of Salmonella enterica serovar Anatum and 81.7% of S. enterica serovar Typhimurium isolates. Resistance to the conventional first-line drugs (ampicillin, chloramphenicol, and cotrimoxazole), cefotaxime and ceftazidime, and ciprofloxacin was found in 32.5 to 49.0%, 20.3 to 20.4%, and 3.2% of isolates, respectively. A total of 76 (3.1%) isolates were resistant to azithromycin, which was associated with mph(A), erm(42), erm(B), and possibly the enhanced expression of efflux pump(s) due to ramAp or defective ramR. mph(A) was found in 53% of the 76 azithromycin-resistant isolates from 11 serovars and located in an IS26-mph(A)-mrx(A)-mphR(A)-IS6100 unit in various incompatibility plasmids and the chromosomes. erm(42) in S. enterica serovar Albany was carried by an integrative and conjugative element, ICE_erm42, and in S. enterica serovar Enteritidis and S. Typhimurium was located in IS26 composite transposons in the chromosomes. erm(B) was carried by IncI1-I(α) plasmids in S. Enteritidis and S. Typhimurium. ramAp was a plasmid-borne ramA, a regulatory activator of efflux pump(s), found in only S. enterica serovar Goldcoast. Since the azithromycin resistance determinants are primarily carried on mobile genetic elements, they could easily be disseminated among human bacterial pathogens. The ramAp-carrying S. Goldcoast isolates displayed azithromycin MICs of 16 to 32 mg/L. Thus, the epidemiological cutoff value of ≤16 mg/L of azithromycin proposed for wild-type NTS should be reconsidered. IMPORTANCE Antimicrobial resistance in NTS isolates is a major public health concern in Taiwan, and the mechanisms of azithromycin resistance are rarely investigated. Azithromycin and carbapenems are the last resort for the treatment of invasive salmonellosis caused by multidrug-resistant (MDR) and extensively drug-resistant Salmonella strains. Our study reports the epidemiological trend of resistance in NTS in Taiwan and the genetic determinants involved in azithromycin resistance. We point out that nearly half of NTS isolates from 2017 to 2018 are MDR, and 20% are resistant to third-generation cephalosporins. The azithromycin resistance rate (3.1%) for the NTS isolates from Taiwan is much higher than those for the NTS isolates from the United States and Europe. Our study also indicates that azithromycin resistance is primarily mediated by mph(A), erm(42), erm(B), and ramAp, which are frequently carried on mobile genetic elements. Thus, the azithromycin resistance determinants could be expected to be disseminated among diverse bacterial pathogens.

describe novel resistance mechanisms, especially in the isolate that was phenotypically resistant but didn't encode any known AMR determinants. The discussion around changing the breakpoints for different serovars is good. Additional subheadings in the results would also help improves the flow of that section. Minor comments: 1. The introduction and discussion are very long. Please consider shortening it by removing information or condensing. Some sentences can be condensed, for example, instead of "in a previous study, we found", say "we previously showed". There are many cases of this and similar examples. 2. Please clarify the significance of azithromycin resistance throughout the manuscript. 3. Please highlight the novel aspects of your work. a. In particular, please clarify which plasmids were novel, and which ones are already published. 4. A heatmap or phylogenetic tree summarising the presence of AMR determinants across the serovars would be a really nice way to convey some of the data rather than relying on large tables. 5. A figure correlating the major AMR determinants with MIC values, and any discrepancies, would greatly enhance the paper. a. Performing statistical analysis on these would further strengthen the paper. 6. Please state in the intro (eg line 91) that azithromycin is a macrolide. 7. Through the discussion, "should" and "would" are used when "could" or "may" are more appropriate -please amend these. 8. Some statements are not very scientific, for example, "are more/less resistant" (eg line 115, 151, 161 and others) needs to be clarified and/or quantified -are you referring to MIC values, the number of AMR genes, or the prevalence of AMR isolates? 9. Line 188-192 -so two copies of floR? Are these paralogs? Are they encoded on the same element? 10. Line 197 -around a dozen -be specific -give the range 11. Line 231 -"relatively different"... ? be specific -how much sequence identity or number of genes shared? Tables & figures: 1. In table 2, I don't think it is accurate to run statistics grouping all other serovars for comparison against the most prevalent ones. Please remove. 2. In table 3, please state that these are percentages, or alternatively, provide the raw values. a. Also, I suggest this table should be supplementary, as these results are implied in table 2. 3. In table 4, please note the MIC breakpoints 4. In table 6, please replace vehicle with compartment a. Also, I suggest this table should be supplementary, and instead to include a summary as a main table b. Further, why are some of the cells in the "Antimicrobial resistance determinant" column blank? 5. Figure 1 should not be a line graph -please replace with a table. 6. Figures 2-5: a. please include a key for the gene colour b. please include a key for the grey shading c. please clarify which are novel sequences and which are previously published d. the "sufflon" was not mentioned in the text -please explain this e. please label boundaries of MGEs such as the ICEs mentioned in the text. Minor English changes: 1. Line 39 -"major public health concern" 2. Line 146 -instinctively > naturally 3. "in recent" > recently Staff Comments:

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Antimicrobial resistance and mechanisms of azithromycin resistance in nontyphoidal Salmonella isolates in Taiwan, 2017-2018; Chiou et al   General comments Chiou et al have presented a detailed overview of AMR in NTS Salmonellae in recent years in Taiwan. They speculate on mechanisms of Azithromycin resistance. The manuscript is detailed however in that detail, the novelty and impact of the study is lost. As azithromycin resistance was only reported in ~3% of isolates, I'm unsure why they focused on this. While the significance is stated in the "importance" section, it is not made very clear throughout the manuscript. There was no effort to describe novel resistance mechanisms, especially in the isolate that was phenotypically resistant but didn't encode any known AMR determinants. The discussion around changing the breakpoints for different serovars is good. Additional subheadings in the results would also help improves the flow of that section.
Minor comments: 1. The introduction and discussion are very long. Please consider shortening it by removing information or condensing. Some sentences can be condensed, for example, instead of "in a previous study, we found", say "we previously showed". There are many cases of this and similar examples.
2. Please clarify the significance of azithromycin resistance throughout the manuscript.
3. Please highlight the novel aspects of your work.
a. In particular, please clarify which plasmids were novel, and which ones are already published.

4.
A heatmap or phylogenetic tree summarising the presence of AMR determinants across the serovars would be a really nice way to convey some of the data rather than relying on large tables. 5. A figure correlating the major AMR determinants with MIC values, and any discrepancies, would greatly enhance the paper.
a. Performing statistical analysis on these would further strengthen the paper.
6. Please state in the intro (eg line 91) that azithromycin is a macrolide.
7. Through the discussion, "should" and "would" are used when "could" or "may" are more appropriate -please amend these.
8. Some statements are not very scientific, for example, "are more/less resistant" (eg line 115, 151, 161 and others) needs to be clarified and/or quantified -are you referring to MIC values, the number of AMR genes, or the prevalence of AMR isolates?
9. Line 188-192 -so two copies of floR? Are these paralogs? Are they encoded on the same element?
10. Line 197 -around a dozen -be specific -give the range 11. Line 231 -"relatively different"… ? be specific -how much sequence identity or number of genes shared? Tables & figures: 1. In 1. Line 39 -"major public health concern" 2. Line 146 -instinctively > naturally 1 Reviewer #1 1. Overall, the manuscript is well-designed and highlights the need for surveillance of azithromycin in Taiwan and worldwide.
2. Lines 16-37: I think some of the methods section needs to be added to the abstract, specifically the phenotypic, PCR, and WGS methods and how many isolates were analysed using these methods. Reply: The methods (AST, PCR, WGS) and the number of isolates tested have been added in the abstract, to be "Antimicrobial resistance was investigated in 2,341 nontyphoidal Salmonella (NTS) isolates recovered from humans in Taiwan between 2017 and 2018 using antimicrobial susceptibility testing. Azithromycin resistance determinants were detected in 175 isolates using polymerase-chain-reaction and confirmed in 81 isolates using whole-genome sequencing" (Lines 16-20 in the revised manuscript).
3. Lines 417-434: There is nothing wrong with the approach taken to sequence and assemble the genomes. However, incomplete and misassemblies can still occur. It would be good to have a paragraph in the results section describing the quality of the assemblies, e.g., the number of contigs, how many contigs were circular, were any plasmid replicons found in chromosomes, and what are the ribosomal chromosome arrangements (these can easily be determined using Socru). Reply: A paragraph has been added in the result section to describe the quality of NGS data, to be "WGS. Whole-genome sequencing using the Illumina sequencing platform was performed on 81 isolates among which 28 from 14 serovars were further sequenced using the Nanopore sequencing platform to investigate resistance genetic determinants and the vehicles for azithromycin resistance. For the isolates with Illumina sequencing data, the median genome coverage depth was 65x (28-125x), the median number of contigs was 117 (65-266), and the median N50 of contigs was 437,365 bp (143,948-757,431 bp). For the 28 isolates with Nanopore sequencing data, the median genome coverage depth was 263x (100-813x), and the median number of circular contigs was 4 (1-8), indicating that the isolates could harbor 0 to 7 plasmids. The sizes of chromosomes of the 28 isolates ranged from 4,645,547 bp to 5,024,703 bp (Table 7). Of the 28 chromosomes, 16 had no resistance genes 2 detected, 2 had an IncQ replicon, and 1 had an IncC replicon" .
Since we want to focus on antimicrobial resistance and the mechanisms of azithromycin resistance, we think it is better not to present the ribosomal chromosome arrangements in the 28 Salmonella isolates in this manuscript.
4. Also were there any discrepancies between the PCR and whole genome sequencing AMR gene results? Reply: The azithromycin resistance genes detected by the PCR are all concordantly identified in the whole genome sequences.
5. Line 23: Instead of using the term "over" I would give the exact percentages.
Reply: The exact percentages have been added to describe the resistance rates, to be "Multidrug resistance was found in 47.3% of all isolates and 96.2% of S. Anatum and 81.7% of S. Typhimurium isolates.
6. Lines 58-61: It would be useful to distinguish which serovars cause typhoid fever and which cause paratyphoid fever. Repy: The serovars that cause typhoid and paratyphoid fever have been indicated, to be "Typhoidal Salmonella serovar, S. Typhi, and paratyphoidal serovars, S. Paratyphi A, S. Paratyphi B, S. Paratyphi C, and S. Sendai, can cause invasive systemic infections in humans and higher primates…" (Lines 63-65).
7. Line 134-139. I understand that you are attempting to categorise the antimicrobials based on how many Salmonella are resistant, but I would still give the exact percentages for each antimicrobial agent. Reply: The description has been revised, to be "Of the 6,861 isolates recovered in 2017-2018, 35.4% were randomly selected for antimicrobial susceptibility testing (Table 1). The susceptibility testing data indicated that 32.5%-49.0% of the isolates were resistant to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, tetracycline, and 3 cotrimoxazole (sulfamethoxazole-trimethoprim), and 20.3%-20.4% were resistant to third-generation cephalosporins (ceftazidime and cefotaxime) ( Table 2)" (Lines 135-140).
8. Line 140. I am assuming by nonsusceptible you mean that they are either resistant or intermediate, but it might be good to define this the first time you use the term.
Reply: The term "nonsusceptible" has been defined when it appears for the first time, to be "nonsusceptible (either resistant or intermediate)" (Line 115).
9. Line 151-155: For this paragraph, I would use actual percentages of isolates belonging to each serovar rather than using the term "most". In addition, I would not use the term "significantly" unless a statistical model was used.
Reply: Thanks for the suggestion. We have revised the paragraph by giving the actual percentages, to be "Among the four most prevalent serovars, S. Anatum had extremely high resistance or nonsusceptibility rates (93.3% to 96.2%) to ampicillin, cefotaxime, ceftazidime, chloramphenicol, ciprofloxacin, cotrimoxazole, streptomycin, sulfamethoxazole, and tetracycline (Table 2). S. Typhimurium also had high resistance rates (45.2% to 83.8%) to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline. In contrast, S.
10. Line 197: Instead of saying around a dozen I would give the range of resistance genes found on these plasmids. Reply: The exact number of resistance genes has been given, to be "…carrying 10 and 12 resistance genes, respectively ( 12. Line 390-393: For the isolates that were whole genome sequenced, were the serovars predicted using PFGE the same as those predicted using whole genome sequencing software, e.g. SISTR. Reply: Yes, the serovars determined via PFGE patterns comparison with those in a Salmonella PFGE database are completely concordant with the serovars predicted from WGS data using the SISTR and SeqSero. 14. Line 429-434. How were insertion sequences and transposons (including ICEs) identified? Reply: Identification of insertion sequences and the ICE_erm42 has been describled in the revised manuscript, to be "Insertion sequences were identified using the tool of ISfinder, which is accessible at the website: https://www-is.biotoul.fr/. The integrative and conjugative element, ICE_erm42 was identified and the mobility of the element was proven in a previous study (23)" (Lines 450-453).
15. Figure 2-5. What software was used to form these alignments and how were the non-AMR gene annotated? Also, many of the alignments have multiple copies of IS26, is this significant? Reply: The software used to make figures 1-4 has been described, as  (42), erm(B), defective ramR, and probably the enhanced expression of efflux pump(s)" to "mph(A), erm (42), erm(B), and possibly the enhanced expression of efflux pump(s) due to ramAp or defective ramR" Reply: Thanks for the suggestion. The sentence has been revised according to the suggestion.
17. Lines 66-67: I would replace the term "first cause" with "largest cause". Reply: Thanks for the correction. It has been revised according to the suggestion.
18. Lines 74-75: I would replace "As the widespread resistance to the conventional first-line drugs among Salmonella serovars" to "Due to the widespread resistance of Salmonella serovars to conventional first-line drugs" Reply: Thanks for the suggestion. The sentence has been revised according to the suggestion.
19. Lines 87-88 I would replace "is inevitable to drive the development of resistance in bacteria" to "selects for resistance to this antimicrobial in bacteria" Reply: Thanks for the suggestion. The sentence has been revised according to the suggestion. 20. Line 89: I would replace "the resistance" with "resistance" 6 Reply: Thanks for the suggestion. It has been revised.
21. Line 91: I would replace "Macrolides express their antibacterial activity" to "Macrolides inhibit bacteria" Reply: Thanks for the suggestion. It has been revised according to the suggestion.
22. Line 99: I would remove "an" Reply: Thanks. The word has been removed.
23. Line 270: I would remove the "as many as" Reply: Thanks. The words have been removed from the sentence.
24. Line 272: "Mph(A)" should not be italicised. Reply: Thanks. The word has been modified.
25. Line 295, 297, 298, 305 and 314: I would replace "is" with "was". Reply: Thanks. The tense of the verbs has been changed to past time.
26. Line 301: I would replace "could move" to "has previously been shown to be able" Reply: Thanks for the suggestion. The sentence has been revised according to the suggestion.
27. Line 318 and 355: I would replace "should" with "could" Reply: Thanks. The word has been replaced.
28. Line 319: I would remove "quite" Reply: Thanks. It has been removed.
29. Line 328: I would replace "thus facilitating the extrusion capacity of "to "extrusion of more" Reply: Thanks for the suggestion. The sentence has been revised according to the suggestion.
30. Line 372: I would replaced "developed through" to "due to" Reply: Thanks. The sentence has been revised. 31. Line 423: Missing closing bracket.
Reply: Thanks for pointing out the error.

Reviewer #3
32. The introduction and discussion are very long. Please consider shortening it by removing information or condensing. Some sentences can be condensed, for example, instead of "in a previous study, we found", say "we previously showed". There are many cases of this and similar examples. Reply: Thanks for the comments. We have revised the wording as possible and removing some information from paragraphs.
33. Please clarify the significance of azithromycin resistance throughout the manuscript. Reply: Thanks for the suggestion. We have addressed the significance of azithromycin resistance in the abstract section as "Azithromycin and carbapenems are the last resort for the treatment of invasive salmonellosis caused by multidrug-resistant (MDR) and extensively drug-resistant Salmonella strains" (lines 41-43), and the introduction section as "While resistance to fluoroquinolones and third-generation cephalosporins is increasing, azithromycin and carbapenems are considered the alternatives for the treatment of invasive salmonellosis caused by MDR and extensively drug-resistant (XDR) Salmonella strains (27-29)" (lines 118-121).
34. Please highlight the novel aspects of your work. a. In particular, please clarify which plasmids were novel, and which ones are already published.
Reply: Thanks for the suggestion. We have emphasized one of the important findings in the Importance section, to be "Our study also indicates that azithromycin resistance is primarily mediated by mph(A), erm(42), erm(B), and ramAp, which are mostly carried on mobile genetic elements. Although the mph(A)-carrying IncHI1A-IncHI1B(pNDM-CIT) and Col(pHAD28)-like plasmids could be reported for the first time in this study, mph(A)-carrying IncHI2-IncHI2A, IncC, and IncFIB(K) plasmids have frequently been found in various species of Enterobacterale and even in Vibrio cholerae" (Lines 48-54).

35.
A heatmap or phylogenetic tree summarising the presence of AMR 8 determinants across the serovars would be a really nice way to convey some of the data rather than relying on large tables. Reply: Thanks for the suggestion. In this study, 81 isolates from 18 serotypes were subjected to whole-genome sequencing, with an average of only 4.5 (1-20) isolates per serovar sequenced. Thus, the data could not be sufficient to demonstrate a good relationship between AMR determinants and serovars. Nevertheless, we are preparing a manuscript that describes the distribution of AMR determinants and plasmids from a large number (558) of S. Typhimurium genomes.
36. A figure correlating the major AMR determinants with MIC values, and any discrepancies, would greatly enhance the paper. a. Performing statistical analysis on these would further strengthen the paper. Reply: Thanks for the suggestion. Because AMR determinants were identified from only 81 isolates, the amount of data may not be sufficient to conduct this type of analysis. However, this is a good point; we may conduct such an analysis in the next study. We have more than 2,000 Salmonella isolates with WGS and MIC data.
37. Please state in the intro (eg line 91) that azithromycin is a macrolide. Reply: Thanks for the suggestion. We have revised the sentence to be "Macrolides, such as azithromycin and erythromycin, inhibit bacteria by…" (Line 97).
38. Through the discussion, "should" and "would" are used when "could" or "may" are more appropriate -please amend these.
Reply: Thanks for the suggestion. We have checked the whole manuscript and revised most of the verbs.
39. Some statements are not very scientific, for example, "are more/less resistant" (eg line 115, 151, 161 and others) needs to be clarified and/or quantified -are you referring to MIC values, the number of AMR genes, or the prevalence of AMR isolates? Reply: Thanks for the comments. We have revised such kind of statement by using the exact numbers or percentages.
40. Line 188-192 -so two copies of floR? Are these paralogs? Are they encoded on the same element? Reply: Yes, the strain harbors two copies of floR in two different genomic islands; one is named SGI1 and the other is an ICE (ICE_erm42).
41. Line 197 -around a dozen -be specific -give the range Reply: Thanks. The sentence has been revised to be "The two S. Typhimurium isolates harbored an additional large IncFIA(HI1)-IncHI1A-IncHI1B or IncC plasmid, carrying 10 and 12 resistance genes, respectively ( were located in the chromosomes, whereas erm (42) and mph(A) in the S. Typhimurium isolate (R18.0292) were carried on an IncI1-I(α) and an IncC plasmid, respectively (Table 7). erm(42) and mph(A ) in S. Enteritidis R17.1476, accompanying with floR, aadA1, and sul3, were located in a 48-kb genomic island flanked by IS26 (Fig. 4). Whereas erm (42)  47. Figure 1 should not be a line graph -please replace with a table. Reply: Thanks. We have presented the data in Figure 1 by a table (Table  3). All the sequences in Figures 1-4 are presented for the first time. Figure 1 presents the sequence alignment of 3 erm(B)-carrying IncI1-I(α) plasmids, Figure 2 presents the sequence aligment of 3 mph(A)-carrying IncFIB(K) plasmids, Figure 3 shows the sequence aligment of 2 mph(A)-carrying transposable elements, and Figure 4 compares the 2 regions of genomic islands with mph(A) and erm (42). Among the 28 isolates with complete genome sequences assembled, S. Albany R17.5974 (harboring an ICE_erm42) and S. Goldcoast R18.0877 (harboring a IncHI2-IncHI2A plasmid with a ramAp) had previously been published; we have stated in the Data Availability, to be "The complete genomic sequences of S.
Albany R17.5974 and S. Goldcoast R18.0877 were assembled and submitted to the NCBI database in previous studies" The "shufflon" has been described briefly in the Result section, to be "The 3 IncI1-I(α) plasmids shared highly similar genetic structures, all carried bla CMY-2 and a clustered inversion region, called shufflon (31)" (Lines 212-213).
We think that only the sequences in Figure 3 could be called IS26 composite transposons because we found tandem repeats of 8 bp at two sides of the IS26 at the ends. IS26 typically generates an 8-bp tandem repeat at the insertion site, as stated "This 22,187-bp unit could be a transposable element as it was flanked by IS6100 and IS26, inserted in a gene encoding a PfkB family carbohydrate kinase, and generated an 8-bp tandem repeat at the insertion site. Similarly, mph(A) and 7 other resistances genes aac(3)-IVa, aadA2, aph(4)-Ia, bla TEM-1 , dfrA12, floR, and sul1, in the S. Typhimurium isolate (R17.3867) were clustered in an 82,497-bp region in the chromosome (Fig. 3). This 82,497-bp genetic unit could be an IS26 composite transposon as it was flanked by IS26 at both ends and generated an 8-bp tandem repeat at the insertion site" (Lines 232-239).
49. Line 39 -"major public health concern" Reply: Thanks for the correction of the usage.
50. Line 146 -instinctively > naturally Reply: Thanks for the correction of the word usage. Thank you for submitting your manuscript to Microbiology Spectrum. When submitting the revised version of your paper, please provide (1) point-by-point responses to the issues raised by the reviewers as file type "Response to Reviewers," not in your cover letter, and (2) a PDF file that indicates the changes from the original submission (by highlighting or underlining the changes) as file type "Marked Up Manuscript -For Review Only". Please use this link to submit your revised manuscript -we strongly recommend that you submit your paper within the next 60 days or reach out to me. Detailed instructions on submitting your revised paper are below.

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